C5aR plus MEK inhibition durably targets the tumor milieu and reveals tumor cell phagocytosis

C5aR antagonist and MEK inhibition combination therapy changes the tumor microenvironment and induces ongoing tumor cell phagocytosis.

Dear Dr. Ratner, Thank you for submitting your manuscript entitled "C5aR antagonism together with MEK inhibition durably targets the tumor microenvironment and reveals ongoing tumor cell apoptosis" to Life Science Alliance.The manuscript was assessed by expert reviewers, whose comments are appended to this letter.We invite you to submit a revised manuscript addressing the Reviewer comments.
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--By submitting a revision, you attest that you are aware of our payment policies found here: https://www.life-sciencealliance.org/copyright-license-feeB. MANUSCRIPT ORGANIZATION AND FORMATTING: Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authorsWe encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** Reviewer #1 (Comments to the Authors (Required)): In this study, Perrino et al investigated the effects of C5aR deficiency or inhibition in combination with MEK inhibition in mouse model of plexiform neurofibromas.They observed that the combination of MEK and C5aR inhibition did not improve the tumor control induced by MEK inhibition alone.However, they observed that C5aR inhibition induced macrophage and SC apoptosis, and other modifications of the tumor microenvironment, including cytokines.Unfortunately, the relevance of these findings is not explored in the study.The study is of potential interest since this tumor is highly infiltrated by C5aR1+ macrophages which may be reactivated in an antitumor phenotype upon C5aR inhibition.However, most results are not explained mechanistically, thus strongly reducing the interest and soundness of the study.Conclusions are not supported by solid experimental evidence.For instance, how is C5aR inhibition inducing macrophage and SC apoptosis?And macrophage-mediated phagocytosis of SC? Are the alterations of the tumor microenvironment observed upon combined treatment relevant in terms of tumor growth and anti-tumor immune responses?
Reviewer #2 (Comments to the Authors (Required)): The authors have identified C5a as a potential target in Plexiform Neurofibromas.Using appropriate genetic and pharmacological inhibition models, the authors have demonstrated that C5aR surface presentation is enhanced in the PNFs and further accentuated in the settings of MEK inhibition.The authors have shown that ablating or inhibiting C5aR leads to enhanced Macrophage and Schwann cell apoptosis and increased Macrophage phagocytosis of Schwann cells.Combined MEK inhibition and C5aR antagonism bring sustained alteration of the tumor microenvironment.This study offers C5aR as a potential new druggable target in the PNFs, for which no durable cure exists.As such, the study is translationally relevant and warrants dissemination to the scientific community.However, I have a few comments to strengthen the output of the manuscript.
1.While the impact of C5aR antagonism on the TME is evident, it needs to be clarified why there was no treatment benefit of the combination treatment with MEKi.The authors have shown that the combination is not toxic and well tolerated; however, if there is no benefit to the tumor outcome, there would be no point in proposing this treatment.How do the authors address this concern?
2. The authors have shown some signs of the durability of the TME-altering response in the combination settings.However, as they suggested, with a longer-term tumor study, it is possible to determine whether the tumor will shrink or continue to enlarge after the treatment.I recommend the authors to address this.
3. In a few instances throughout the manuscript, the authors have stated facts in a way that, while NOT FALSE, could BE Misleading to the readers.For example, on page 17, the title of the last paragraph is "Longer term treatment of C5aRA+MEKi reduces tumor size".While this statement is factually absolutely correct, the true message that should have been communicated was that the combination is NOT doing any better than the MEKi alone.After all, the authors are not trying to demonstrate the impact of MEKi in this manuscript but rather the benefit of the combination.The authors must modify this and others (wherever applicable) to reflect the true essence of their experimental data.We thank the reviewers and the editor for their comments on our manuscript "C5aRantagonism in combination with MEK inhibition durably targets the tumor micro-environment and reveals ongoing tumor cell phagocytosis", by Perrino, Ahmari and co-workers.You invited us to submit a revised manuscript addressing the Reviewer comments (8/21/23).We appreciate that this revision took longer than your typical 3-month turnaround, but Reviewer 2 requested an in vivo study that required significant analysis.We addressed the reviewer's comments point by point and highlighted the changes in yellow in the manuscript.We hope that you will find this revised manuscript appropriate for publication in Life Science Alliance.

Reviewer #1
In this study, Perrino et al investigated the effects of C5aR deficiency or inhibition in combination with MEK inhibition in mouse model of plexiform neurofibromas.They observed that the combination of MEK and C5aR inhibition did not improve the tumor control induced by MEK inhibition alone.However, they observed that C5aR inhibition induced macrophage and SC apoptosis, and other modifications of the tumor microenvironment, including cytokines.Unfortunately, the relevance of these findings is not explored in the study.The study is of potential interest since this tumor is highly infiltrated by C5aR1+ macrophages which may be reactivated in an antitumor phenotype upon C5aR inhibition.However, most results are not explained mechanistically, thus strongly reducing the interest and soundness of the study.Conclusions are not supported by solid experimental evidence.
For instance, how is C5aR inhibition inducing macrophage and SC apoptosis?And macrophage-mediated phagocytosis of SC? Response: This is indeed an important question and indeed, a question we have tried to address.We show using mutant mice that phagocytosis by tumour macrophages is dependent on C5aR and independent of MEK inhibition.We do not yet know what causes cell death, but we characterized cells which die in tumors and show that at least dying Schwann cells are phagocytosed by macrophages in a MEK or C5AR inhibitor-dependent fashion (Figure 5).
To make these points more clearly, we thoroughly revised Figure5 and the accompanying test (Pages 19-20).
Are the alterations of the tumor microenvironment observed upon combined treatment relevant in terms of tumor growth and anti-tumor immune responses?

Response:
We spent considerable effort to answer this important question, and the data is presented in a new Figure 7.The answer is no, the combined therapy does not cause durable effects on tumor growth.Regarding immune cells, despite the abundance of C5aR1+ myeloid cells in PNF, very few changes occur on dendritic cells or macrophages on blocking this receptor.However, in the off-treatment groups, we identified significant changes in the tumor microenvironment that persist for at least 4 months, especially after single agent C5aRantagonism (new Figures 7B & 7C).
Reviewer #2 (Comments to the Authors (Required)): The authors have identified C5a as a potential target in Plexiform Neurofibromas.Using appropriate genetic and pharmacological inhibition models, the authors have demonstrated that C5aR surface presentation is enhanced in the PNFs and further accentuated in the settings of MEK inhibition.The authors have shown that ablating or inhibiting C5aR leads to enhanced Macrophage and Schwann cell apoptosis and increased Macrophage phagocytosis of Schwann cells.Combined MEK inhibition and C5aR antagonism bring sustained alteration of the tumor microenvironment.This study offers C5aR as a potential new druggable target in the PNFs, for which no durable cure exists.As such, the study is translationally relevant and warrants dissemination to the scientific community.However, I have a few comments to strengthen the output of the manuscript.
1.While the impact of C5aR antagonism on the TME is evident, it needs to be clarified why there was no treatment benefit of the combination treatment with MEKi.The authors have shown that the combination is not toxic and well tolerated; however, if there is no benefit to the tumor outcome, there would be no point in proposing this treatment.How do the authors address this concern?

Response:
We completely agree with this reviewer.Although a promising target initially, our data indicate that as a single agent or combination with MEK inhibition C5aR antagonism insufficient to drive therapeutic benefit; combinations or alternatives will be necessary.Given the importance of this idea, we have added following sentence to the discussion "Even four months after stopping treatment, tumors persisted (Figure 7), yet immune cell changes persisted.There was a notable and prolonged change in tumor microenvironment in the durability study.These effects best correlated decreased numbers of CD8 T cells……."Overall, although a promising target initially, our data indicate that as a single agent or combination with MEK inhibition, C5aR antagonism is insufficient to drive therapeutic benefit in PNF; thus, additional combinations or alternative schedules require investigation."(Page 28).
2. The authors have shown some signs of the durability of the TME-altering response in the combination settings.However, as they suggested, with a longer-term tumor study, it is possible to determine whether the tumor will shrink or continue to enlarge after the treatment.I recommend the authors to address this.

Response:
We agreed with this reviewer and had initiated a longer durability study before manuscript submission.We show that data as an entirely new Figure 7.We found that tumors do not shrink after transient drug exposure.However, A8 Δ71-73 .driven C5AaR1 antagonism is sufficient to modulate the PNF tumor microenvironment for months (Pages 22-23).
3. In a few instances throughout the manuscript, the authors have stated facts in a way that, while NOT FALSE, could BE Misleading to the readers.For example, on page 17, the title of the last paragraph is "Longer term treatment of C5aRA+MEKi reduces tumor size".While this statement is factually absolutely correct, the true message that should have been communicated was that the combination is NOT doing any better than the MEKi alone.After all, the authors are not trying to demonstrate the impact of MEKi in this manuscript but rather the benefit of the combination.The authors must modify this and others (wherever applicable) to reflect the true essence of their experimental data.

Response:
We replaced this section heading to read: + MEKi reduces tumor size, similar to MEKi alone."(Page 17) 4. On page 3, line 60: it would be "tumor mass" not "tumor cells"

Response:
We did not change this, as the tumor mass is contributed by tumor cells, immune cells, stromal cells, and matrix, and because 30% of cells are macrophages, as a percent of all tumor cells.5. Page 16, line 360: it would be "...significantly increased apoptotic cell..." not "...significantly increased dead cell..."

Response:
Given that recent literature finds that not only apoptotic cell death but also other types of cell death can result in cleavage of DNA by Casp3 (e.g.pyroptosis) we prefer this nomenclature.This now reads: "At this early time point, MEK inhibition alone had little effect on death of cells isolated from murine PNF.In contrast, genetic deletion of C5aR1 significantly increased CC3+ cells (Fig. 3C), and the combination of C5aR1 deletion and MEK inhibition did not alter the amount of cell death" (Page 16).
Additionally, we modified figure 2H by adding more time points and sample numbers.We reanalyzed the data using two-way ANOVA, and modified supplemental figures 4 and 5. Thank you for submitting your revised manuscript entitled "C5aR plus MEK inhibition durably targets the tumor miliu and reveals tumor cell phagocytosis".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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Thank you for submitting your Research Article entitled "C5aR plus MEK inhibition durably targets the tumor milieu and reveals tumor cell phagocytosis".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.